The central nervous system (CNS) requires a tightly controlled environment free of toxins and pathogens to provide the proper chemical conditions for synaptic transmission. This environment is maintained by the 'blood brain barrier' (BBB), which is composed of highly specialized blood vessels whose endothelium display specialized tight junctions and unusually low rates of transcellular vesicular transport (transcytosis). While BBB breakdown has recently been associated with various neurological disorders, an intact BBB also poses a major obstacle for drug delivery to the CNS. Pharmaceutical companies spend billions of dollars to develop drugs that can penetrate the BBB to treat disease. However, little progress has been made on manipulating the BBB due to a significant knowledge gap in understanding how BBB function is regulated and identifying the essential molecular constituents governing its processes. This limited understanding has also thwarted our ability to therapeutically manipulate the BBB. The major impediment to understanding the BBB is identifying its essential constituent and unraveling the mechanism by which these key regulators control BBB function. However, the current in vitro models rely on fully differentiated endothelial cells, which already contain unique properties that prevent their use in reconstitution studies. Similarly, the main technique to study the BBB has been EM, however its static snapshots do not provide information on active and dynamic vesicular transport, directionality, or their specifi routes to allow investigators to interrogate the key molecular mechanisms that regulate BBB integrity. Recently, with our research background in developmental co-wiring of nervous and vascular systems, we used the traditional developmental approach, to first mapped the precise timing of BBB formation and then identified neural cues that induce CNS endothelium to acquire BBB properties, and molecules with possible roles in